A vaccine to combat malaria is a highly desirable public health tool to reduce morbidity and mortality in African children. In order to achieve this goal it will be important to gain a detailed understanding of the impact of malaria on the generation and maintenance of immunological memory. This project represents a collaborative effort between Dr. Pierce, Dr. Louis Miller, Dr. Peter Crompton and with scientists at the Malaria Research and Training Center (MRTC) at the University of Mali. Over the last year we have focused our efforts on gaining an understanding of the generation, maintenance and activation of immunological memory in response to natural malaria infection. In humans, B cell memory is encoded both in long-lived memory B cells and in plasma cells that reside in the bone marrow. The mechanisms by which memory B cells or plasma cells are generated and maintained over a life time are not known. Current evidence, primarily from serological epidemiological studies, indicates that immunological memory to malaria is slow to be acquired, incomplete and short lived. Thus, despite chronic exposure to Plasmodium falciparum (Pf) from birth from infectious mosquito bites, children in endemic areas do not acquire immunity that protects them from severe disease until the age of five. Consequently, children under five years of age are susceptible to severe disease that accounts for nearly one million deaths each year in Africa alone. Acquisition of immunity that protects against severe disease but not against mild disease is acquired prior to adolescence and an immunity sufficient to prevent disease but not to eliminate parasites is acquired only in adolescence to early adulthood. Our current hypothesis is that Pf infections disrupt the normal mechanisms by which memory is generated, maintained or activated. To study the acquisition of immunity to malaria we initiated a longitudinal study on a cohort of 225 volunteers, 2-25 years of age in Kambila, a village outside of Bamako, the capital city of Mali in June 2006 prior to the malaria transmission season which runs July through December. Transmission is also highly intense in this area ensuring that all individuals in the cohort will be infected at least once with malaria during the transmission season. This highly seasonal transmission with six months of intense malaria exposure and six months free of malaria offers a near ideal condition to evaluate the impact of malaria infection on the generation and maintenance of malaria immunity. Our approach was to measure a variety of immune parameters in the peripheral blood of individuals prior to the malaria season, 7 and 14 days after their first attack of clinical malaria, at 2 month intervals through the transmission season and at the end of the dry season. The immune parameters measured before the season were correlated with clinical malaria yes or no. The immune parameters in samples 7 and 14 days after an attack of clinical malaria were analyzed to determine the impact of malaria on these parameters. The samples at the end of the dry season were analyzed to determine the longevity of any changes in immune parameters acquired during the transmission season. Our analysis of the acquisition of malaria-specific MBCs in the Kambila cohort showed that Pf-specific memory B cells increased with age but only in a gradual stepwise fashion over years of exposure to malaria. We observed that the number of malaria-specific MBCs transiently increased after the first malaria infection. The increase in MBCs was accompanied by a short term rise in the levels of malaria-specific antibody. Of significant interest we also observed a transient increase in the number of MBCs specific for an antigen unrelated to malaria, namely tetanus toxoid (TT) following malaria infections. However, the increase in TT-specific MBCs was not accompanied by an increase in the level of TT-specific antibody. Together these novel results suggest that MBCs are maintained in response to nonspecific innate immune system stimulants that accompany infection but the differentiation of MBCs to PCs only occurs in response to antigen. Although classical antigen-specific MBCs were generated in response to malaria infections we also observed a large increase in the number of atypical FcRL4+ MBCs. FcRL4 was first described to mark a unique population of MBCs in healthy individuals in mucosal lymphoid tissues in close proximity to epithelial boundaries in the body, site of pathogen invasion. These FcRL4+ MBCs had distinctive functional characteristics and gene expression profile. Because of their location FcRL4+ tissue MBCs are thought to play a role in antibody responses to pathogens. Subsequently FcRL4+ MBCs were identified in large numbers in the peripheral blood of HIV-infected viremic individuals suggesting that FcRL4+ MBCs may differentiate in response to chronic antigenic stimulation. We found that FcRL4+ MBCs could represent up to 30% of B cells in Malian children as young as two years of age. We have initiated studies to determine when and how atypical MBCs are generated and their function. Understanding the origin and function of these atypical MBCs should be of significant interest in the development of malaria vaccines. Over the last year we also completed studies to assess the nature of the antibody response to Pf in our Kambila cohort in collaboration with Dr. P. Felgner (U.C. Irvine) using a proteome chip containing approximately 25% of the Pf proteome. Our results indicate that the intensity and complexity of the malaria-specific antibody response increased with age and that the intensity and complexity of the serum antibodies at the beginning of the malaria season predicts protection from malaria. We also observed that the complexity and intensity of antibodies transiently rose during the season but most of the increases were lost by six months and did not predict protection. We also identified an antibody signature associated with malaria immunity. Antibodies specific for a subset of 49 proteins were found to be significantly increased in immune children as compared to nonimmune children. The increased antibody responses specific for the top five of these 49 proteins proved to be a highly sensitive and specific indicator of malaria immunity. Studies are underway to validate the findings in additional cohorts that differ in the genetic background of the volunteers and in the malaria transmission patterns. We have also initiated studies to test the efficacy of these signature proteins as vaccine candidates in animal models of malaria. Lastly, we are developing a microarray containing Var gene products to be used to search for an antibody signature associated with severe malaria. In June of this year we officially closed our study site in Kambila but will continue to analyze immune parameters in samples collected in 2007, 2008 and 2009. At the same time we initiated site preparation for a new prospective cohort study of naturally acquired malaria immunity in Kalifabougou, including carrying out a pilot study to estimate the burden and distribution of Pf malaria;improving the Kalifabougou clinic and building a residence for study personnel. The new study of 1,500 individuals will allow us to validate findings from the Kambila study and to approach a number of additional questions concerning the relationship between Pf and the immune system in the acquisition of malaria immunity.